Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The biogenesis of multivesicular bodies (MVBs) is topologically equivalent to virion budding. Hence, a number of viruses exploit the MVB pathway to build their envelope and exit from the cell. By expression of dominant negative forms of Vps4 and Vps24, two components of the MVB pathway, we observed an impairment in infectious herpes simplex virus (HSV) assembly/egress, in agreement with a recent report showing the involvement in HSV envelopment of Vps4, the MVB-specific ATPase (C. M. Crump, C. Yates, and T. Minson, J. Virol. 81:7380-7387). Furthermore, HSV infection resulted in morphological changes to MVBs. Glycoprotein B (gB), one of the most highly conserved glycoproteins across the Herpesviridae family, was sorted to MVB membranes. In cells expressing the dominant negative form of Vps4, the site of intracellular gB accumulation was altered; part of gB accumulated as an endoglycosidase H-sensitive immature form at a calreticulin-positive compartment, indicating that gB traffic was dependent on a functional MVB pathway. gB was ubiquitinated in both infected and transfected cells. Ubiquitination was in part dependent on ubiquitin lysine 63, a signal for cargo sorting to MVBs. Partial deletion of the gB cytoplasmic tail resulted in a dramatic reduction of ubiquitination, as well as of progeny virus assembly and release to the extracellular compartment. Thus, HSV envelopment/egress and gB intracellular trafficking are dependent on functional MVB biogenesis. Our data support the view that the sorting of gB to MVB membranes may represent a critical step in HSV envelopment and egress and that modified MVB membranes constitute a platform for HSV cytoplasmic envelopment or that MVB components are recruited to the site(s) of envelopment.
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PMID:Intracellular trafficking and maturation of herpes simplex virus type 1 gB and virus egress require functional biogenesis of multivesicular bodies. 1768 35

A protocol for purification of the two-subunit complex of herpes simplex virus type 1 (HSV-1) helicase-primase by metal affinity chromatography is presented. In order to bind the enzyme complex consisting of UL5 and UL52 gene functions to the affinity column, the C-terminus of the UL5 gene of HSV-1 strain ANG was fused in-frame with a sequence encoding six histidines, resulting in a His6-tagged DNA helicase (UL5his) when expressed via recombinant baculovirus. In addition, hybridoma cell lines producing anti-UL5 IgG were generated for screening of DNA helicase expression. Initial purification trials revealed that the presence of low concentrations of imidazole in the wash buffers interfered with the binding of the helicase-primase subunit complex to the metal affinity resin. Alternative means, such as high salt, altered pH, and substitution of imidazole by histidine tetrapeptide (His4), were tested. From those, the addition of His4 in combination with an acidic pH turned out to be very efficient for the removal of protein contaminants from a Ni2+-NTA (nitrilotriacidic acid) affinity resin. By applying only one column step, the present protocol yields a helicase-primase preparation, which is suitable for inhibitor screening and further functional studies. The final preparation is free of interfering enzyme activities, and exerts each of the enzymatic functions described for a two subunit complex, i.e., DNA-dependent ATPase, DNA primase, and DNA helicase activities.
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PMID:One-step column purification of herpes simplex virus 1 helicase-primase subcomplex using C-terminally his-tagged UL5 subunit. 1939 88

The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4DN) in HCMV replication. Vps4DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4DN or CHMP1DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.
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PMID:Human cytomegalovirus exploits ESCRT machinery in the process of virion maturation. 1964 Sep 81

Herpes simplex virus type 1 (HSV-1) acquires its mature virus envelope by budding into the lumen of cytoplasmic membranous compartments carrying the viral glycoproteins. In a cellular context, a budding process with identical topology occurs during the formation of intraluminal vesicles in multivesicular bodies. The cellular machinery that mediates this budding process is composed of four protein complexes termed endosomal sorting complexes required for transport (ESCRTs) and several associated proteins, including the ATPase VPS4. We have recently shown that functional VPS4 is specifically required for the cytoplasmic envelopment of HSV-1. We now demonstrate that, consistent with a role of VPS4 in virus envelopment, dominant-negative ESCRT-III proteins potently block HSV-1 production. Retroviruses are known to recruit the ESCRT machinery by small peptide motifs termed late domains. These late domains interact with various ESCRT components and thereby promote ESCRT recruitment. The best-characterized late-domain interacting ESCRT proteins are ALIX and TSG101. The presence of potential ALIX and TSG101 binding sequence motifs in various structural HSV-1 proteins suggested a functional role of these proteins in HSV-1 envelopment. We therefore used a set of dominant-negative proteins, as well as RNA interference, to characterize the contribution of ALIX and TSG101 to HSV-1 production. Interestingly, despite the strict requirement for a functional ESCRT-III complex, our data suggest that HSV-1 production is independent of ALIX and TSG101 expression. In line with these data, we also find that ESCRT-III proteins and VPS4A/B are specifically incorporated into mature HSV-1 virions.
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PMID:Herpes simplex virus type 1 production requires a functional ESCRT-III complex but is independent of TSG101 and ALIX expression. 1969 79

The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and UL8. We generated each of the potential two-subunit complexes, UL5-UL52, UL5-UL8, and UL52-UL8, and used a series of kinetic and photo-cross-linking studies to provide further insights into the roles of each subunit in DNA binding and primer synthesis. UL8 increases the rate of primer synthesis by UL5-UL52 by increasing the rate of primer initiation (two NTPs --> pppNpN), the rate-limiting step in primer synthesis. The UL5-UL8 complex lacked any detectable catalytic activity (DNA-dependent ATPase, primase, or RNA polymerase using a RNA primer-template and NTPs as substrates) but could still bind DNA, indicating that UL52 must provide some key amino acids needed for helicase function. The UL52-UL8 complex lacked detectable DNA-dependent ATPase activity and could not synthesize primers on single-stranded DNA. However, it exhibited robust RNA polymerase activity using a RNA primer-template and NTPs as substrates. Thus, UL52 must contain the entire primase active site needed for phosphodiester bond formation, while UL5 minimally contributes amino acids needed for the initiation of primer synthesis. Photo-cross-linking experiments using single-stranded templates containing 5-iodouracil either before, in, or after the canonical 3'-GPyPy (Py is T or C) initiation site for primer synthesis showed that only UL5 cross-linked to the DNA. This occurred for the UL5-UL52, UL5-UL52-UL8, and UL5-UL8 complexes and whether the reaction mixtures contained NTPs. Photo-cross-linking of a RNA primer-template, the product of primer synthesis, containing 5-iodouracil in the template generated the same apparent cross-linked species.
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PMID:Herpes simplex virus-1 helicase-primase: roles of each subunit in DNA binding and phosphodiester bond formation. 1978 34

Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.
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PMID:Inhibition of herpes simplex virus type 1 replication by adeno-associated virus rep proteins depends on their combined DNA-binding and ATPase/helicase activities. 2010 23

The protein binding to the origin of replication of the herpes simplex virus type 1 (HSV-1) is DNA helicase encoded by the UL9 gene of the herpes virus. The protein specifically binds to two binding sites in the viral DNA replication origins OriS or OriL. In order to determine the role of the UL9 protein in the initiation of replication and find efficient inhibitors of the UL9 activity, we have synthesized a recombinant UL9 protein expressed in E. coli cells. It was found that the recombinant UL9 protein binds to Boxes I and II in the OriS and possesses the DNA helicase and ATPase activities. In a complex with a fluorescent analog of ATP, two molecules of the ATP analog bind to one protein dimer molecule. It was also found that the UL9 protein in the dimer form can bind simultaneously to two DNA fragments, each containing specific binding sites for the protein. The interaction of the recombinant UL9 protein with the 63-mer double and single-stranded oligonucleotides OriS and OriS* has been investigated, which correspond to the origin of replication of herpes simplex virus. From the titrations of OriS and OriS* by ethidium bromide in the presence and absence of the UL9 protein, the equilibrium affinity constants of the protein binding to OriS and OriS* have been determined. A DNase I footprinting study showed that bis-linked netropsin derivatives exhibit preferences for binding to the AT-cluster in the origin of replication OriS and inhibit the fluctuation opening of AT-base pairs in the AT-cluster. The drugs also prevent the formation of an intermediate conformation of OriS* that involves a disordered tail at the 3'-end and stable Box I-Box III hairpin to which the UL9 helicase selectively binds. The stabilization by bis-netropsins of the AT-rich hairpin at its 3' end can inhibit the helicase activity. It was concluded that the antiviral activity of bis-netropsins may be associated with the inhibitory effects of bis-netropsins on these two stages of the reaction catalyzed by helicase UL9.
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PMID:[Complex of the herpes simplex virus initiator protein UL9 with DNA as a platform for the design of a new type of antiviral drugs]. 2042 77

The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5' to 3' helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.
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PMID:Herpes simplex virus type 1 helicase-primase: DNA binding and consequent protein oligomerization and primase activation. 2106 46

Herpes simplex virus 1 (HSV-1) capsids leave the nucleus by a process of envelopment and de-envelopment at the nuclear envelope (NE) that is accompanied by structural alterations of the NE. As capsids translocate across the NE, transient primary enveloped virions form in the perinuclear space. Here, we provide evidence that torsinA (TA), a ubiquitously expressed ATPase, has a role in HSV-1 nuclear egress. TA resides within the lumen of the endoplasmic reticulum (ER)/NE and functions in maintaining normal NE architecture. We show that perturbation of TA normal function by overexpressing torsinA wild type (TAwt) inhibits HSV-1 production. Ultrastructural analysis of infected cells overexpressing TAwt revealed reduced levels of surface virions in addition to accumulation of novel, double-membrane structures called virus-like vesicles (VLVs). Although mainly found in the cytoplasm, VLVs resemble primary virions in their size, by the appearance of the inner membrane, and by the presence of pUL34, a structural component of primary virions. Collectively, our data suggest a model in which interference of TA normal function by overexpression impairs de-envelopment of the primary virions leading to their accumulation in a cytoplasmic membrane compartment. This implies novel functions for TA at the NE.
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PMID:A functional role for TorsinA in herpes simplex virus 1 nuclear egress. 2177 50

Alzheimer's disease susceptibility genes, APP and gamma-secretase, are involved in the herpes simplex life cycle, and that of other suspect pathogens (C. pneumoniae, H. pylori, C. neoformans, B. burgdorferri, P. gingivalis) or immune defence. Such pathogens promote beta-amyloid deposition and tau phosphorylation and may thus be causative agents, whose effects are conditioned by genes. The antimicrobial effects of beta-amyloid, the localisation of APP/gamma-secretase in immunocompetent dendritic cells, and gamma secretase cleavage of numerous pathogen receptors suggest that this network is concerned with pathogen disposal, effects which may be abrogated by the presence of beta-amyloid autoantibodies in the elderly. These autoantibodies, as well as those to nerve growth factor and tau, also observed in Alzheimer's disease, may well be antibodies to pathogens, due to homology between human autoantigens and pathogen proteins. NGF or tau antibodies promote beta-amyloid deposition, neurofibrillary tangles, or cholinergic neuronal loss, and, with other autoantibodies, such as anti-ATPase, are potential agents of destruction, whose formation is dictated by sequence homology between pathogen and human proteins, and thus by pathogen strain and human genes. Pathogen elimination in the ageing population and removal of culpable autoantibodies might reduce the incidence and offer hope for a cure in this affliction.
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PMID:Alzheimer's Disease: APP, Gamma Secretase, APOE, CLU, CR1, PICALM, ABCA7, BIN1, CD2AP, CD33, EPHA1, and MS4A2, and Their Relationships with Herpes Simplex, C. Pneumoniae, Other Suspect Pathogens, and the Immune System. 2225 44


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